Fluorescent neutralization and adherence inhibition assays

ABSTRACT

The present invention comprises rugged, inexpensive, reliable, and sensitive laboratory assays of antibody-based viral neutralization activity and antibody-based viral adherence inhibition activity. The assays use inactivated, fluorescently-labeled virus, allowing the tests to be performed without extensive safety precautions. The interaction of the labeled virus with target cells is monitored using flow cytometric methods. A preferred embodiment uses simple and inexpensive flow cytometry methodologies and equipment, such as bead array readers used as simplified flow cytometers. The assays are rapid, taking no longer than a few hours and are readily conducted by a trained technician. The assays are sensitive because they use labeled viruses at low concentrations and determine neutralizing and blocking capacity of sera and antibody at low concentrations. The methods are appropriate for high-throughput screening of large panels of samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S. PatentApplication No. 61/113,263, filed Nov. 11, 2008, which is herebyincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The ability of an anti-viral pathogen vaccine to produce an effectiveantibody response is typically evaluated in various types of virusneutralization tests or assays. Common features of such tests includemonitoring the level of infectivity of the virus (natural or attenuated)in a standardized target cell culture, and evaluating the reduction ininfectivity of the virus after incubation with the tested serum/sera orantibody solution(s) of interest.

The dilution of a serum or antibody (Ab) solution that provides 50% ormore reduction of infectivity is referred to as the ‘neutralizationtiter’ (Niedrig et al. (2008) Clin. Vaccine Immunol. 15, 177; Guidelinesfor Plaque-Reduction Neutralization Testing of Human Antibodies toDengue Viruses, WHO, 2007). Virus neutralization assays are widely usedworldwide for a variety of viruses, from the relatively common, such asinfluenza and herpes simplex, to the most feared and dangerous viruses,such as smallpox, yellow fever, and dengue hemorrhagic fever (Niedrig etal. (2008) Clin. Vaccine Immunol. 15, 177; Guidelines forPlaque-Reduction Neutralization Testing of Human Antibodies to DengueViruses, WHO, 2007).

In the virology community, the plaque reduction neutralization test(PRNT) is generally considered the gold standard of neutralizationassays for studying anti-viral humoral immune responses (Niedrig et al.(2008) Clin. Vaccine Immunol. 15, 177; Guidelines for Plaque-ReductionNeutralization Testing of Human Antibodies to Dengue Viruses, WHO, 2007;Roukens et al. (2008) PLoS ONE, 3, e1993; Niedrig et al. (1999) Trop.Med. Int. Health 4, 867). In the PRNT, a highly diluted virus culture,with or without various concentrations of a test serum or an antibodysolution, is added to a culture of a confluent layer of target cells,and the level of infectivity is measured by the number of cell-freelacunas appearing in the culture after a period of incubation (typicallya few days).

Currently, various more recent immunosorption methods compete with thePRNT, such as the immunofluorescent assay (IFA) (Kraus et al. (2007), J.Clin. Microbiol. 45, 3777; Niedrig et al. (1999) Trop. Med. Int. Health4, 867; Groot & Riberiro (1962) Bull. WHO 27, 699; Vazquez et al. (2003)J. Virol. Methods 110, 179; Barry et al. (1991) Am. J. Trop. Med. Hyg.44, 79; Deubel et al. (1983) Am. J. Trop. Med. Hyg. 32, 565)), where theinfectivity level of virus is evaluated using fluorescently-labeled,virus-specific antibodies applied to fixed samples of target cells afterincubation with live virus and washing. The IFA, although sensitive andvirus-specific, remains under consideration, because the antibody titersfrom the IFA often do not correlate well with those from the PRNT(Niedrig et al. (1999) Trop. Med. Int. Health 4, 867; Groot & Riberiro(1962) Bull. WHO 27, 699; Vazquez et al. (2003) J. Virol. Methods 110,179; Barry et al. (1991) Am. J. Trop. Med. Hyg. 44, 79; Deubel et al.(1983) Am. J. Trop. Med. Hyg. 32, 565).

A microneutralization method similar to the IFA, but using enzyme-linkedimmunosorption, was developed in Centers for Disease Control andPrevention (CDC). In this assay, the level of virus infectivity with orwithout tested sera is estimated by measuring nuclear protein (NP) ofthe avian influenza virus expressed in the target MDCK cells by stainingthe permeabilized fixed cells with the NP-specific monoclonal Ab labeledwith horseradish peroxidase (HRP) (Rowe et al. (1999) J. Clin.Microbiol. 37, 937-943).

Another direct microneutralization (MN) assay is also practiced by theWorld Health Organization (WHO). In the MN assay, a confluent layer oftarget cells is infected with a diluted virus culture either in thepresence or absence of a test serum/sera or antibody solution(s) ofinterest, and the rate of virus reproduction is evaluated by measuringreleased virus concentrations with a standard hemagglutination assay(HA) technique. The assay is described in the “WHO Manual on AnimalInfluenza Diagnosis and Surveillance,” (WHO/CDS/CSR/NCS/2002.5 Rev. 1)and may be found at the website beginning with “www.” and ending with“who.int/vaccine_research/diseases/influenza/WHO_manual_on_animal-diagnosis_and_surveillance_(—)2002_(—)5.pdf”.The protocol is simple and straightforward, although use of the HAtechnique presents certain limitations to the sensitivity of the method.

In general, the PRNT, IFA and MN have several complicating features:

-   -   Use of live virus raises safety and personnel protection issues.    -   Incubation of the target cells with live virus often requires        significant time (up to ˜5-7 days) to allow for infection to        properly develop and infectivity to be reliably evaluated,        making the tests lengthy and cumbersome.    -   PRNT and IFA include multi-step fixation and staining protocols.

The use of modern flow cytometry techniques promises to significantlyimprove existing neutralization assays. In several published studies,viral infection of target cells in the presence or absence of a testserum/sera or antibody solution(s) of interest has been monitored byflow cytometric methods, where viruses or their cell-expressed proteincomponents were stained with fluorescent tags (Kremser et al. (2004)Anal. Chem. 80, 7360; Sliva et al. (2004) Virol. J. 1, 14; You et al.(2006) Int. J. Nanomedicine 1, 59; Nichols et al. (1993) Arch. Virol.130, 441; Klingen et al. (2008) J. Virol. 82, 237; Lonsdale et al.(2003) J. Virol. Meth. 110, 67-71; Wang et al. (2004) J. Virol. Meth.120, 207-215; Collins & Buchholz (2005) J. Virol. Meth. 128, 192-197).For example, permeabilized target cells were stained with fluorescentantibodies specific to target-expressed viral proteins (Lonsdale et al.(2003) J. Virol. Meth. 110, 67-71), and green fluorescent protein (GFP)was incorporated in a recombinant vector and expressed in infected cells(You et al. (2006) Int. J. Nanomedicine 1, 59; Wang et al. (2004) J.Virol. Meth. 120, 207; Collins & Buchholz (2005) J. Virol. Meth. 128,192; Earl et al. (2003), J. Virol. 77, 10684). Other examples includedirect labeling of the virus (Kremser et al. (2004) Anal. Chem. 80,7360; Klingen et al. (2008) J. Virol. 82, 237). Such labeling can beperformed via direct chemical tagging with fluorochromes (Kremser et al.(2004) Anal. Chem. 80, 7360), or electrostatic attachment of quantumdots to the envelope proteins of the virus (Sliva et al. (2004) Virol.J. 1, 14). Nichols et al. (1993; Arch. Virol. 130, 441) and Klingen etal. (2008; J. Virol. 82, 237) developed an ingenious virus stainingmethod, growing the virus on a pre-stained target cells.

These flow cytometric methods, however, also use live viruses orrecombinant vectors, raising safety and other issues similar to thoselisted for PRNT and IFA assays. These methods, while sensitive andinformative in the research setting, can hardly be consideredappropriate as routine high-throughput assays. Thus, there is acontinuing need for rugged, reliable, and sensitive laboratory methodsfor microneutralization assays.

SUMMARY OF THE INVENTION

Addressing the problems with existing assays, the present inventioncomprises a rugged, reliable, and sensitive laboratory method for avirus neutralization assay, characterized by the following:

-   -   Use of inactivated, fluorescently-labeled virus, allowing the        tests to be performed without extensive safety precautions.    -   Interaction of the labeled virus with the target cells,        monitored by flow cytometric methods. A preferred embodiment        uses the simplest and least expensive flow cytometry        methodologies and equipment. A more preferred embodiment uses a        bead array reader, such as a BioPlex, as a simplified flow        cytometer.    -   The assay is rapid, taking no longer than a few hours (normally,        ˜1.5-4 h) and is readily conducted by a trained technician.    -   The assay is sensitive; that is, it uses labeled viruses at low        concentrations and measures blocking/neutralizing capacity of        sera and antibodies at low concentrations.    -   The assay is appropriate for automation and high-throughput        screening of sera and culture fluids.    -   The assay is inexpensive, using, for example, the rugged BioPlex        bead array platform as a simplified flow cytometer at a cost        ˜20% of a regular flow cytometer such as, for example, a BD LSR        II (BD Biosciences).

Embodiments of the present invention comprise affinity fluorescentlabeling of the virus used in the fmNt (fluorescence-based microneutralization) assay. For example, the virus is sparsely labeled withbiotinylated virus-specific antibody possessing low neutralizingcapacity, and streptavidin-phycoerythrin conjugate is attached to thebiotins. This method can work equally well labeling live or inactivatedvirus, in pure culture or one containing high levels of contaminants. Inanother embodiment, the inactivated virus is sparsely biotinylated, andstreptavidin-phycoerythrin conjugate is attached to the biotins.

In another embodiment, for example, a bead array reader, such as aBioPlex, is used as a simplified flow cytometer to detect fluorescenceof the labeled virus engulfed by, or attached to, target cells. Using abead array reader, such as a BioPlex, instead of a flow cytometer canreduce the cost of the assay by ˜5-fold and allows working with lowernumbers of target cells in the sample.

Another embodiment of the present invention involves “addressed”affinity quenching of the phycoerythrin fluorescence using ananti-phycoerythrin antibody coupled with the QSY-9 quenching dye. Thismethod increases the efficiency of quenching surface-bound fluorescence,which is undesirable in fluorescence-based microneutralization (fmNt)experiments.

Another embodiment of the present invention comprises a “FluorescentAdherence Inhibition Assay” (fADI), a method to measure the capacity ofa virus-specific antibody or anti-virus sera to block adherence of thevirus to the surface of target cells. The method comprises a combinationof a hemagglutination inhibition assay (HAI) and fluorescencemicroneutralization assay and features at least about a 10-foldimprovement in sensitivity versus previous fluorescence-basedmicroneutralization assays (fmNt) and hemagglutination inhibition assays(HAI).

The present invention is also directed to the following specificembodiments. In a first embodiment, the invention is directed to amethod for determining a neutralizing activity of a test antibody,comprising:

a) incubating a test antibody with a fluorescently-labeled virus to forma mixture,

b) incubating a population of target cells with the mixture of a) underconditions permitting endocytosis of the labeled virus by the targetcells,

c) measuring fluorescence of labeled virus endocytozed by the targetcells, and

d) comparing the fluorescence measured in c) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining a neutralizing activity of a test antibody.

In a second embodiment, the present invention is directed to a methodfor determining a neutralizing activity of a test antibody, comprising:

a) incubating a test antibody with a fluorescently-labeled virus to forma mixture,

b) incubating a population of target cells with the mixture of a) underconditions permitting endocytosis of the labeled virus by the targetcells,

c) incubating the population of target cells of b) with a quencher offluorescence of labeled virus bound to the surface of the cells,

d) measuring fluorescence of labeled virus endocytozed by the targetcells, and

e) comparing the fluorescence measured in d) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining a neutralizing activity of a test antibody.

In one aspect, the quencher of fluorescence of the labeled virus boundto the surface of the cells is a protease. In another aspect, thequencher of fluorescence of the labeled virus bound to the surface ofthe cells is an antibody that specifically binds the fluorescent labelof the labeled virus, wherein the antibody is conjugated to at least onequenching compound, and wherein the quenching compound is a fluorescentquenching dye. In a preferred aspect, the labeled virus is a virushaving a biotinylated antibody bound thereto wherein the antibody isconjugated to a phycoerythrin:streptavadin conjugate, and the antibodythat specifically binds the fluorescent label of the labeled virus is aphycoerythrin-specific antibody conjugated to quenching dye QSY-9,quenching dye QSY-21, or both quenching dyes. The quenching dye may alsobe trypan blue or crystal violet.

In a third embodiment, the present invention is directed to a method fordetermining a neutralizing activity of a test antibody, comprising:

a) incubating a test antibody with a fluorescently-labeled virus to forma mixture,

b) incubating a population of target cells with the mixture of a) underconditions permitting endocytosis of the labeled virus by the targetcells,

c) incubating the population of target cells of b) with a quencher offluorescence of labeled virus bound to the surface of the cells,

d) staining the population of target cells of c) with a dye,

e) measuring fluorescence of labeled virus endocytozed by the targetcells, and

f) comparing the fluorescence measured in e) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining a neutralizing activity of a test antibody.

In one aspect, the quencher of fluorescence of the labeled virus boundto the surface of the cells is a protease. In another aspect, thequencher of fluorescence of the labeled virus bound to the surface ofthe cells is an antibody that specifically binds the fluorescent labelof the labeled virus, wherein the antibody is conjugated to at least onequenching compound, and wherein the quenching compound is a fluorescentquenching dye. In a preferred aspect, the labeled virus is a virushaving a biotinylated antibody bound thereto wherein the antibody isconjugated to a phycoerythrin:streptavadin conjugate, and the antibodythat specifically binds the fluorescent label of the labeled virus is aphycoerythrin-specific antibody conjugated to quenching dye QSY-9,quenching dye QSY-21, or both quenching dyes. In a further aspect, thequencher is dye, and the dye is trypan blue or crystal violet.

In another aspect, the population of cells is stained with a dye havinga weak red and infrared fluorescence to facilitate classification of thecells in a BioPlex bead array reader. Alternatively, the population ofcells is stained with a dye that quenches the fluorescent label of thelabeled virus. In certain embodiments, the dye serves both functions. Ina specific aspect, the staining dye is trypan blue or crystal violet.

In a fourth embodiment, the present invention is directed to a methodfor determining an inhibitory activity of a test antibody, comprising:

a) incubating a test antibody with a fluorescently-labeled virus to forma mixture,

b) incubating a population of target cells with the mixture of a) underconditions subduing endocytosis and permitting cell surface adherence ofthe labeled virus to the target cells,

c) measuring fluorescence of labeled virus adhered to the surface of thetarget cells, and

d) comparing the fluorescence measured in c) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining an inhibitory activity of a test antibody.

In a fifth embodiment, the present invention is directed to a method fordetermining an inhibitory activity of a test antibody, comprising:

a) incubating a test antibody with a fluorescently-labeled virus to forma mixture,

b) incubating a population of target cells with the mixture of a) underconditions subduing endocytosis and permitting cell surface adherence ofthe labeled virus to the target cells,

c) staining the population of target cells of b) with a dye,

d) measuring fluorescence of labeled virus adhered to the surface of thetarget cells, and

e) comparing the fluorescence measured in d) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining an inhibitory activity of a test antibody.

In one aspect, the population of cells is stained with a dye having aweak red and infrared fluorescence to facilitate classification of thecells in a BioPlex bead array reader. Alternatively, the population ofcells is stained with a dye that quenches the fluorescent label of thelabeled virus. In certain embodiments, the dye serves both functions. Ina specific aspect, the staining dye is trypan blue or crystal violet.

In each of the embodiments of the invention, the labeled virus may be avirus selected from the group consisting of adenoviruses, filoviruses,flaviviruses, herpesviruses, poxviruses, parvoviruses, reoviruses,picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses,retroviruses, and hepadnaviruses, conjugated to a fluorescent label.

In a preferred aspect, the labeled virus comprises an influenza virusconjugated to a fluorescent label. In an equally preferred aspect, thelabeled virus comprises an influenza A virus conjugated to a fluorescentlabel. In a further preferred aspect the labeled virus comprises an H1N1influenza virus or H3N2 influenza virus or H5N1 influenza virusconjugated to a fluorescent label. In an additionally preferred aspectthe labeled virus is Marburg hemorrhagic fever virus-like particle orgamma-inactivated Ebola virus conjugated to a fluorescent label. In eachaspect, the fluorescent label may be phycoerythrin or allophycocyanin.

Alternatively, in each of the embodiments of the invention, the labeledvirus may be a biotinylated virus selected from the group consisting ofadenoviruses, filoviruses, flaviviruses, herpesviruses, poxviruses,parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses,rhabdoviruses, retroviruses, and hepadnaviruses, conjugated to astreptavadin:fluorescent label. In a preferred aspect, the labeled viruscomprises a biotinylated influenza virus conjugated to astreptavadin:fluorescent label. In an equally preferred aspect, thelabeled virus comprises a biotinylated influenza A virus conjugated to astreptavadin:fluorescent label. In a further preferred aspect, thelabeled virus comprises a biotinylated H1N1 influenza virus or H3N2influenza virus or H5N1 influenza virus conjugated to astreptavadin:fluorescent label. In an alternative aspect, the labeledvirus comprises biotinylated Marburg virus-like particles (VLP) taggedwith streptavidin-phycoerythrin conjugate. In another alternativeaspect, the labeled virus comprises gamma-inactivated and biotinylatedEbola virus tagged with streptavidin-phycoerythrin conjugate. In eachaspect, the fluorescent label may be phycoerythrin or allophycocyanin.

In addition, in each of the embodiments of the invention, the labeledvirus may be a virus selected from the group consisting of adenoviruses,filoviruses, flaviviruses, herpesviruses, poxviruses, parvoviruses,reoviruses, picornaviruses, togaviruses, orthomyxoviruses,rhabdoviruses, retroviruses, and hepadnaviruses, bound by a fluorescentlabel:streptavadin-conjugated biotinylated antibody that specificallybinds the virus. In a preferred aspect, the labeled virus comprises aninfluenza virus bound by a biotinylated antibody that specifically bindsthe virus and that has a fluorescent label:streptavadin conjugate boundthereto. In an equally preferred aspect, the labeled virus comprises aninfluenza A virus bound by a biotinylated antibody that specificallybinds the virus and that has a fluorescent label:streptavadin conjugatebound thereto. Such antibodies include the biotinylated anti-influenza AH1 specific antibody #1307 and the biotinylated antibody is thebiotinylated anti-influenza A H3 specific antibody #1317. In a furtherpreferred aspect, the labeled virus comprises an H1N1 influenza virus orH3N2 influenza virus H5N1 influenza virus bound by a biotinylatedantibody that specifically binds the virus and that has a fluorescentlabel:streptavadin conjugate bound thereto. In an alternative aspect,the labeled virus comprises betapropiolactone (BPL)-inactivated ‘avian’influenza H5N1 virus bound by a biotinylated antibody that specificallybinds the virus and that has a fluorescent label:streptavadin conjugatebound thereto. In an additional aspect, the labeled virus is Marburghemorrhagic fever virus-like particle or gamma-inactivated Ebola virusbound by a biotinylated antibody that specifically binds the virus andthat has a fluorescent label:streptavadin conjugate bound thereto. Ineach aspect, the fluorescent label may be phycoerythrin orallophycocyanin.

In each of the embodiments of the invention, the virus may be aninactivated or an attenuated virus. When inactivated, the virus may beinactivated using BPL, or UV or gamma irradiation. In an alternativeaspect, the virus inactivation method can be any inactivation methodthat preserves the ability of the virus to adhere specifically to targetcells.

In each of the embodiments of the invention, the population of targetcells may be a mammalian cell line, an avian cell line, an amphibiancell line, or other cell line susceptible to viral attack. In oneaspect, the population of target cells comprises a human cell line. Inanother aspect, the population of target cells comprises avianerythrocytes. In a further aspect, the population of target cellscomprises a cell line selected from the group consisting of Madin-Darbycanine kidney epithelial cells and Vero green monkey kidney epithelialcells.

In each of the embodiments of the invention, measuring of fluorescencemay be through the use of a flow cytometer or a bead array reader. Forexample, a BioPlex-100, a BioPlex-200, a Luminex-100, or a Luminex-200bead array reader may be used.

In each of the embodiments of the invention, the incubating of thetarget cells with the labeled virus may be at 4° C. or 37° C.

In each of the embodiments of the invention, the neutralizing activityof sera or an antibody is the blocking of entry of the labeled virusinto the target cells.

In each of the embodiments of the invention, the blocking activity ofsera or an antibody is blocking adherence of the labeled virus to thetarget cells, or blocking entry of the labeled virus into the targetcells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Relative sizes of the affinity SA-PE (streptavidin:phycoerythrinconjugate) fluorescent probe and influenza virus. The affinity labelrepresents a high-affinity biotinylated virus-specific antibody labeledwith SA-PE fluorescent conjugate. All dimensions are shown approximatelyproportional to the natural size.

FIG. 2. Testing replacement of the affinity label by an anti-influenzaserum: ELISA experiment. Solomon Islands H1N1 BPL-inactivated virus, CDCstandard, was labeled in the ELISA wells with biotinylatedanti-influenza A H1N1 antibody (ViroStat #1307), and tagged with SA-PE.An attempt was made to replace the label (SA-PE conjugated biotinylatedViroStat #1307) with an excess of anti-influenza serum from a vaccinatedhigh-responding blood donor (#419). FIG. 2A=incubation at roomtemperature. FIG. 2B=incubation at 37° C. “Virus,serum”=virus+replacement sera; “Virus, no serum”=virus+PBS; “Blank,serum”=no virus+replacement sera; “Blank, no serum”=no virus+PBS.

FIG. 3. Testing replacement of the affinity label by an anti-influenzaserum: BioPlex experiment. Luminex beads coated with a recombinant H1hemagglutinin from Solomon Islands H1N1 virus (“Sol. Isl. H1”) or NewCaledonia H1N1 virus (“New Caled. H1”) were stained with thebiotinylated ViroStat #1307 antibody coupled with the SA-PE conjugateand then incubated with an excess of anti-influenza serum from avaccinated high-responding donor (“Serum”) or PBS (“No Serum”). FIG.3A=incubation at 4° C. FIG. 3B=incubation at 37° C.

FIG. 4. Quenching of phycoerythrin fluorescence using ananti-phycoerythrin antibody coupled with QSY quencher. MDCK (Madin-Darbycanine kidney) cells were labeled using a biotinylated anti-LDLantibody, coupled with the SA-PE conjugate. The cells were incubated at4° C. with an anti-PE (phycoerythrin) antibody tagged with QSY-9fluorescence quenching dye. Trypan blue was added (400 μg/mL) to achieveadditional non-specific quenching.

FIG. 5. Staining cells with trypan blue made them ‘readable’ in theBioPlex bead array reader. Depicted is the two-dimensionalclassification panel of the BioPlex-100, the y-axis representinginfrared fluorescence, and the x-axis, red fluorescence. Multiple paleovals in the middle of the map are classification regions designated forthe appropriately coded beads. Upper panel: Unstained MDCK cells wereregistered by the BioPlex, but their fluorescence was not read, becausethey were not classified as legitimate objects. Middle and Lower panels:Cells were stained with 500 μg/mL trypan blue. Their images shiftedupward and rightward in the classification panel. Many cells wereclassified as legitimate objects, and their fluorescence was measured bythe BioPlex reader. The lower, smaller circle on each panel encompassesnormal healthy cells; the upper, larger circle on each panel encompassesdamaged and dying cells.

FIG. 6. Comparing fluorescence of the labeled cells measured in the flowcytometer and the BioPlex bead array reader. Regions 11, 17, and 25 ofthe BioPlex classification panel (upper left) were used to calculate thepooled average mean fluorescence index (MFI) of the labeled cells readin the BioPlex. The table shows numbers of cells registered in each ofthe three regions and the mean fluorescence index (MFI) calculated.

FIG. 7. Layout and the protocol for the BioPlex fmNt experiment withBPL-inactivated Solomon Islands H1N1 virus and MDCK target cells. Twoidentical plates were prepared in which the labeled virus or thecomponents of the labeling without virus was incubated with MDCK cellsat 4° C. or at 37° C. In each plate, the left part of the layout wasprepared without the PE-specific quencher, and the right parts with thePE-specific quencher. The concentrations/dilutions of the componentsindicated are prior to mixing in the wells. The detailed description ofthe procedure is in the text.

FIG. 8. Results of the proof-of-concept fmNt experiment performed at 37°C. Components of the label without virus did not bring fluorescence tothe target cells (“SA-PE alone” and “Anti-H1:Btn:SA-PE”). Together withthe virus (H1N1 Solomon Islands, BPL-inactivated), the fluorescence ofthe cells increased dramatically (“Sol. Isl. H1+anti-H1:Btn:SA-PE”).Pre-incubation of the virus with anti-influenza serum strongly reducedthe fluorescence (“Sol. Isl. H1 blocked with anti-Fluserum+anti-H1:Btn:SA-PE”). PE-specific (anti-PE:QSY-9) and non-specific(TB; trypan blue) fluorescence quenchers reduced the fluorescence fromsurface-bound virus.

FIG. 9. Results of the proof-of-concept fmNt experiment performed at 4°C. Fluorescent signals of the target cells as well as the relativeeffects of the fluorescence quenchers increased, because at the lowertemperature the endocytosis is subdued, and most of the viruses remainedattached to the surface of the cells.

FIG. 10. Components, layout, and protocol for measuring neutralizingcapacity of commercial anti-influenza A antibodies.

FIG. 11. Neutralizing capacity of commercial anti-influenza antibodies,as determined using the fmNt method. Dashed lines show the fitting tothe theoretical titration curves and the 50% cut-off titers. The titersshow moderate-to-low neutralizing capacity for both ViroStat andMillipore specimens. FIG. 11A=AB #1301; FIG. 11B=AB #1074. All sampleswere read in duplicate.

FIG. 12. Neutralizing capacity of human anti-influenza sera, asdetermined using the fmNt method. The fmNt titers shift sharply upwardafter vaccination thus demonstrating a strong neutralizing capacity ofthe post-vaccination sera. FIG. 12A=Donor #355, pre-vaccination; FIG.12B=Donor #355, post-vaccination; FIG. 12C=Donor #419, pre-vaccination;FIG. 12D=Donor #419, post-vaccination. All samples were read induplicate.

FIG. 13. Comparison of the neutralizing titers of pre- andpost-vaccination sera from donors immunized against seasonal influenzaobtained using the VaxDesign fmNt protocol (FIG. 13A) and CDC (FIG. 13B)and WHO (FIG. 13C) MN protocols. The data from the fmNt experiments withBPL-inactivated Solomon Islands virus demonstrated a strong correlationwith the experiments using live virus according to the standard MNprotocols practiced by CDC and WHO. The fmNt experiment demonstrated thesuperior sensitivity of the assay of the present invention.

FIG. 14. Comparison of fmNt (FIG. 14A) and fADI (FIG. 14B) protocols.The fADI protocol required almost 50% less incubation time and did notuse an anti-PE surface quencher. Affinity labeled Solomon Islands H1N1BPL-inactivated virus; MDCK target cells.

FIG. 15. Comparison of fmNt and fADI measurements. FIG. 15A: Comparisonof fluorescence of the target cells in the fmNt and fADI experiments inthe presence (“AB #1301”) or absence (“No AB”) of the polyclonalanti-influenza A antibody #1301, ViroStat. FIG. 15B: Fluorescence of thetarget cells in the fADI experiment at different concentrations of thevirus. The level of fluorescence acceptable in the BioPlex-assistedexperiment shown as a dashed line corresponds to significantly higherdilutions of the virus than in the fmNt experiment. Affinity-labeledSolomon Islands H1N1 BPL-inactivated virus; MDCK target cells.

FIG. 16. Comparison of fmNt and fADI titers for a commercialanti-influenza A antibody. Upper insert: fmNt and fADI titratingneutralizing capacity of anti-influenza A, ViroStat #1301. Lower insert:Juxtaposition of neutralizing titers of the ViroStat #1301 antibody andthe virus dilutions used in the fmNt and fADI experiments. Highersensitivity of the fADI method corresponds to the higher dilution of thevirus used in the experiment. Affinity-labeled Solomon Islands H1N1BPL-inactivated virus; MDCK target cells.

FIG. 17. Neutralizing capacity of human anti-influenza sera towards H1N1virus, as determined using the fADI method and turkey erythrocytes astargets. Sera samples were pre-diluted according to their pre-determinedHAI titers. The fADI titers shift upward after vaccination. FIG.17A=Donor #608, pre-vaccination; FIG. 17B=Donor #608, post-vaccination;FIG. 17C=Donor #145, pre-vaccination; FIG. 17D=Donor #145,post-vaccination. All samples were in duplicate. Affinity-labeled NewCaledonia H1N1 BPL-inactivated virus; turkey erythrocytes as targetcells.

FIG. 18. Correlation of fADI titers and traditional HAI titers for thepanel of human sera. Standard HAI experiments with human group Oerythrocytes and BPL inactivated New Caledonia H1N1 virus. Turkeyerythrocytes were used as target cells.

FIG. 19. Neutralizing capacity of human anti-influenza sera towards H3N2virus, as determined using the fADI method and turkey erythrocytes astargets. Sera samples were pre-diluted according to their pre-determinedHAI titers. The fADI titers shift upward after vaccination. FIG.19A=Donor #608, pre-vaccination; FIG. 19B=Donor #608, post-vaccination.All samples were in duplicate. Affinity-labeled Wisconsin H3N2BPL-inactivated virus; turkey erythrocytes were used as target cells.

FIG. 20. Correlation of fADI titers and traditional HAI titers for thepanel of human sera. Standard HAI experiments with human group Oerythrocytes and BPL inactivated Wisconsin H3N2 virus. Affinity-labeledWisconsin H3N2 BPL-inactivated virus; turkey erythrocytes were used astarget cells.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above in the Summary, the invention is primarily directedto two assays. In the first, second and third embodiments, the inventionis directed to methods for determining the neutralizing activity of atest antibody. In the fourth and fifth embodiments, the invention isdirected to methods for determining the inhibitory activity of a testantibody.

In the methods for determining the neutralizing activity of a testantibody, fluorescently-labeled virus is prepared or obtained, and afirst portion of the labeled virus is incubated with serum/sera orantibody solution(s) of interest (the “test antibody”). A second portionof the labeled virus is incubated with a positive or negative control,such as an antibody that is known to bind (or not bind) the labeledvirus or no antibody at all. A population of target cells are thenprepared and incubated with the mixture of labeled virus and antibodyunder conditions permitting endocytosis of the labeled virus by thetarget cells. After a period of time, fluorescence of the labeled virusthat has been endocytozed by the target cells is measured. Comparison ofthe fluorescence measured in the population of cells exposed to thelabeled virus-test antibody against the fluorescence measured in thepopulation of cells exposed to the labeled virus-control provides anindication of the ability of the test antibody to inhibit penetration ofthe target cells by the labeled virus, and thus the neutralizingactivity of a test antibody.

The second and third embodiments of the invention provide aspects of theinvention that reduce background fluorescence in the assay. In thesecond embodiment, after the population of cells is incubated with themixture of labeled virus and antibody under conditions permittingendocytosis, the target cells are incubated with a quencher thatquenches the fluorescence of any labeled virus that remains bound to thesurface of the cells. In this manner, the signal produced by labeledvirus that has been internalized can be more readily distinguished fromthe background signal produced by labeled virus that remains on thesurface of the cell.

The third embodiment of the invention adds the additional step ofstaining the population of target cells with a dye. The dye facilitatesclassification of the cells in a BioPlex bead array reader, or the dyequenches the fluorescent label of the labeled virus. In certainembodiments, the dye serves both functions. When facilitatingclassification of the cells, the dye has a weak red and infraredfluorescence. Preferred dyes include trypan blue and crystal violet.

The fourth and fifth embodiments of the invention are similar to thefirst, second and third, but in contrast to assaying for endocytosis ofthe labeled virus into a target cells, these latter embodiment aredirected to methods for determining the ability of a test antibody toblock adherence of the virus to the surface of the cell. In the fourthembodiment, fluorescently-labeled virus is prepared or obtained, and afirst portion of the labeled virus is incubated with serum/sera orantibody solution(s) of interest (the “test antibody”). A second portionof the labeled virus is incubated with a positive or negative control,such as an antibody that is known to bind (or not bind) the labeledvirus or no antibody at all. A population of target cells are thenprepared and incubated with the mixture of labeled virus and antibodyunder conditions that subdue endocytosis and permit cell surfaceadherence of the labeled virus to the target cells. After a period oftime, fluorescence of the labeled virus that has adhered to the surfaceof the target cells is measured. Comparison of the fluorescence measuredin the population of cells exposed to the labeled virus-test antibodyagainst the fluorescence measured in the population of cells exposed tothe labeled virus-control provides an indication of the ability of thetest antibody to inhibit binding by the labeled virus to the surface ofthe target cells, and thus the inhibitory activity of a test antibody.

The fifth embodiment of the invention adds the additional step ofstaining the population of target cells with a dye. The dye facilitatesclassification of the cells in a BioPlex bead array reader, or the dyequenches the fluorescent label of the labeled virus. In certainembodiments, the dye serves both functions. When facilitatingclassification of the cells, the dye has a weak red and infraredfluorescence.

As used in the various embodiments and aspects of the invention, thequencher of surfaced-localized labeled virus may be any means thatquenches surface fluorescence alone, without reducing the fluorescenceof internalized labeled virus. Suitable quenchers include proteasesspecific for the fluorescent label. In a preferred aspect, the quencheris an antibody that specifically recognizes and binds to the fluorescentlabel of the virus. Such antibodies are conjugated to at least onequenching compound. As indicated above, suitable quenching compoundsinclude dyes, such as quenching dye QSY-9 (Invitrogen) and quenching dyeQSY-21. The antibodies may be conjugated to more than one dye.

There are few limitations of the identity of the virus that may be usedin the embodiments and aspects of the invention. For example, the virusmay be selected from among the adenoviruses, filoviruses, flaviviruses,herpesviruses, poxviruses, parvoviruses, reoviruses, picornaviruses,togaviruses, orthomyxoviruses, rhabdoviruses, retroviruses, andhepadnaviruses. In a preferred aspect, the virus comprises an influenzavirus, such as an influenza A virus. In a equally preferred aspect thevirus comprises the H1N1 influenza virus or H3N2 influenza virus or H5N1‘avian’ influenza virus. In another preferred aspect, the viruscomprises Marburg virus-like particles (VLPs) or gamma-inactivated Ebolavirus.

The label that is used to produce the labeled viruses of the presentinvention is preferably a fluorescent label. As taught herein, threemeans of labeling a virus with a fluorescent label have been developed.In a first aspect, the virus may be directly labeled by conjugating afluorescent label to a virus, using chemical means known in the art,such as chemical linking. In a second aspect, the virus may bebiotinylated and then labeled with a streptavadin:fluorescent labelconjugate. Many medically important biotinylated viruses are alsocommercially available. In a third aspect, the virus may be labeledusing fluorescently-labeled antibody that specifically recognizes andbinds the virus. Such antibodies may be directly tagged by conjugating afluorescent label to the antibody, using chemical means known in theart, such as chemical linking. Alternatively, the anti-virus antibodiesmay be biotinylated and then labeled with a streptavadin:fluorescentlabel conjugate. As with the virus, many biotinylated antibodies arecommercially available. Suitable antibodies include the biotinylatedanti-influenza A H1 specific antibody #1307 (ViroStat) and thebiotinylated anti-influenza A H3 specific antibody #1317 (ViroStat).

In each of the embodiments of the invention, the virus may be a livevirus, an inactivated virus, or an attenuated virus. When inactivated,the virus may be inactivated using BPL, or UV or gamma irradiation, orby any other chemical or physical method that preserves the ability ofthe virus to adhere specifically to the target cells.

In each of the embodiments of the invention, the population of targetcells may be any cell line that can be bound by a virus, and/or intowhich a virus can penetrate. Suitable cell lines include mammalian celllines, avian cell lines, amphibian cell lines, and other cell linessusceptible to viral attack. In one aspect, the population of targetcells comprises a human cell line. In another aspect, the population oftarget cells comprises avian erythrocytes. In a further aspect, thepopulation of target cells comprises a cell line selected from the groupconsisting of Madin-Darby canine kidney epithelial cells and Vero greenmonkey kidney epithelial cells.

In each of the embodiments of the invention, fluorescence may bemeasured through the use of a flow cytometer or a bead array reader. Forexample, a BioPlex-100, a BioPlex-200, a Luminex-100, or a Luminex-200bead array reader may be used.

In each of the embodiments of the invention, the various steps in thedisclosed methods can be conducted at different temperatures. Forexample, the step of incubating target cells with the labeled virus mayconducted at temperatures conducive or inhibitory to endocytosis, suchas at 37° C. or 4° C.

In each of the embodiments of the invention, the neutralizing activityof sera or an antibody is the blocking of entry of the labeled virusinto the target cells.

In each of the embodiments of the invention, the fluorescent label maybe phycoerythrin or allophycocyanin, or any other fluorescent moleculeor molecular complex detectable via flow cytometry.

EXAMPLES

Materials: Suitable cells, viruses, biological materials, and equipmentfor conducting the examples described here include the following:

Cells: MDCK cell line (ATCC, #CCL-34); Vero cell line (ATCC); Turkeyblood in citrate buffer, Rockland Immunochemicals.

Viruses: Solomon Islands H1N1 BPL-inactivated influenza virus; NewCaledonia H1N1 BPL-inactivated influenza virus; Wisconsin H3N2BPL-inactivated influenza virus. All viruses can be obtained asBPL-inactivated standards from the Centers for Disease Control, Atlanta,Ga.

Antibodies: Rabbit anti-human LDL, R & D Systems, #BAF2148; Goatanti-R-phycoerythrin, Rockland Immunochemicals # 600-101-387; Goatanti-influenza A H1 IgG, ViroStat #1301; Goat anti-influenza A H1IgG:biotin, ViroStat #1307; Goat anti-influenza A H3 IgG:biotin,ViroStat #1317; Goat anti-influenza A H1 IgG, Millipore #ab1074.

Other components: Streptavidin-phycoerythrin, Millipore, #45-001;EZ-Link Sulfo-NHS-LC-biotin, Pierce #21335; Amine-reactive quenching dyeQSY-9, Invitrogen #Q-20131; Human anti-influenza sera, from FloridaBlood Bank, as used at VaxDesign for testing anti-influenza vaccinationresponses in the year 2008; Bovine serum albumin, heat-shock separated,low endotoxin, Sigma-Aldrich #A9430; Chicken egg albumin, grade V,Sigma-Aldrich #A5503; Human serum albumin, Sigma-Aldrich #A8763.

Equipment: Bead array readers: BioPlex-100 and BioPlex-200 (BioRad) wereused effectively as simplified flow cytometers; flow cytometer: BD LSRII (BD Biosciences); orbital digital shakers: VWR #97006-944; 96-wellU-shaped plates, clear polystyrene: VWR #29445-154.

Example 1 Influenza Virus as a Model for Developing the NeutralizationAssay: Affinity Fluorescent Labeling

BPL-inactivated influenza virus standards of various strains are readilyavailable, for example, from the US Centers for Disease Control andPrevention (CDC). Solomon Islands H1N1, New Caledonia H1N1, andWisconsin H3N2 strains containing ˜10 ⁹ viral particles/mL were used inmost of the experiments, as examples.

Experiments were conducted with various labels, including directbiotinylation, quantum dots and fluorescent nanoparticles. Affinitylabeling with biotinylated influenza A-specific antibodies withsubsequent attachment of the fluorescent streptavidin-phycoerythrin(SA-PE) conjugate provided the brightest labeling of the influenza Avirus. Direct biotinylation of the virus also provided an acceptablelevel of signal.

It was previously found that the goat polyclonal anti-influenza A H1specific antibody # 1301 (ViroStat) had a high affinity for H1N1viruses, but low neutralizing capacity, when compared with the sera ofinfluenza-vaccinated donors (data not shown). The biotinylated versionof the same antibody (ViroStat # 1307) was used for labeling SolomonIslands H1N1 and New Caledonia H1N1 viruses, while the biotinylatedH3-specific antibody (ViroStat #1317) was used for labeling WisconsinH3N2 virus. Labeling was performed at a low concentration of theantibodies, to prevent significant modification of the virus surface bythe label.

An Example of Labeling Procedure

In embodiments of the present invention, an aliquot of theBPL-inactivated influenza virus, CDC standard, was typically diluted1:10 in PBS containing 0.1% high-grade ovalbumin and 0.1% sodium azide(NaN₃). A biotinylated anti-influenza virus antibody, ViroStat #1307 or#1317 was added to the virus sample on ice, with stirring, to a finalconcentration 2.3 μg/mL. The sample was incubated overnight at 4° C. ona planetary shaker at 600 rpm. Afterwards, phycoerythrin-streptavidinconjugate (SA-PE) was added to the reaction mix on ice with constantstirring to a final dilution of 1:20 in the sample. After a further 24-hincubation at 4° C., the sample was diluted 1:10 in PBS containing 0.1%ovalbumin and 0.1% NaN₃, wrapped in aluminum foil, and stored in therefrigerator until further use. The preparation showed high stabilityfor more than 3 months.

Labeling with a non-neutralizing antibody and SA-PE fluorescentconjugate proved to be facile and reliable, and provided the brightestfluorescence. Such labeling could be applied to inactivated and liveviruses alike, with minimal effect from the contaminants in the virusculture.

From the relative sizes of the virus and the label, sparse surfacelabeling of the virus, even with a tag of high molecular weight, such asan antibody coupled with a SA-PE conjugate, was not expected tosignificantly alter adherence of the virus to target cells or tointerfere with subsequent endocytosis of the virus by the target cells(FIG. 1).

Example 2 Assessing Replacement of the Affinity Label by OtherVirus-Specific Antibodies

An important question needed to be addressed before using the describedaffinity labeling in the neutralization experiments. Testing theneutralizing capacity of a test sera or other fluids requires incubatingthe labeled virus with a significant excess of other virus-specificantibodies in the tested sample, many of which may be cross-reactivewith the viral epitopes recognized by the labeling antibody. It wastherefore important to check whether other virus-specific antibodieswould displace the labeling antibodies on the virus surface.

Label replacement tests were performed at the same concentrations,volumes, temperatures, and times of incubation as the prospectiveneutralization assays (FIG. 2). In the first test, four wells of theELISA plates were coated with inactivated Solomon Islands virus(“Virus”), and the other four wells were left blank (“Blank”), asnegative controls. After blocking with 2% BSA and washing, the wellswere then filled with the labeling biotinylated anti-influenza Aantibody (ViroStat #1307), at 2 μg/mL. After incubation for 2 h at 4° C.and washing, the wells were filled with SA-PE conjugate, at 4 μg/mL.After the second 30-min incubation at 4° C. and washing, the wells werefilled with PBS, and the plates were read in a Synergy HT plate reader(BioTek) in the phycoerythrin fluorescence channel (“0 hours”). Then,the wells were re-filled with either PBS (“no serum”) or high-responderpost-vaccination anti-influenza serum (“serum”, #1250) at a dilution of1/200 (estimated level of anti-influenza IgG, ˜5-10 μg/mL). One of theplates was incubated at room temperature (FIG. 2A), and the other at 37°C. (FIG. 2B). After the first hour of incubation, the plates werewashed, filled with PBS, and read again (“1 hour”), and then re-filledwith the serum and PBS, incubated for 2 h, washed, and read again (“3hours”). No label replacement was detected.

Another setup was performed using Luminex beads, chemically decoratedwith recombinant H1 hemagglutinins from Solomon Islands and NewCaledonia H1N1 viruses, according to a modified Bio-Rad protocol (can befound at the Bio-Rad website www.bio-rad.com) (FIG. 3). The H1-coatedbeads were first incubated with the labeling biotinylated anti-influenzaantibody (ViroStat #1307) in the same conditions as described above forthe ELISA setup. After staining with SA-PE and washing, the beads werefurther incubated with either PBS (“No Serum”), or a high-responderanti-influenza serum (“No Serum”; #1250) at a dilution of 1:200. Then,the beads were washed and read in the BioPlex in the regular multiplexmode (FIG. 3). No replacement was detected.

The results shown in FIGS. 2 and 3 demonstrated that within the testedranges of incubation times and temperatures, no replacement of thelabeling antibodies took place. Insignificant decrease in thefluorescence signal observed for the ELISA samples after the firstincubation, with or without the overlaying anti-influenza serum alike,was caused by washing out of a portion of loosely attached virus.

Some other tests of the replacement of the labeling antibody wereperformed; all of them showed negative results. Especially interestingwas a test with the anti-influenza A H1 (ViroStat #1301), which isactually the same antibody as used for the labeling, although notbiotinylated. In this experiment, the overlay of antibody #1301contained 45 μg/mL of the antibody (i.e., 20 times higher than was usedin the labeling with Ab #1307). Nonetheless, no replacement of the labelwas observed after a 1-h incubation at 37° C. (data not shown).

Example 3 Quenching of Surface-Bound Phycoerythrin Fluorescence

Monitoring fluorescently labeled virus engulfed by target cellsinevitably requires subduing the fluorescence of surface-adherent virus.This can be done via extensive protease treatment of the cell surface,or (preferably) quenching of the surface-bound fluorescence using non-or low-fluorescence quenching agents. Trypan blue (TB) and crystalviolet have been used successfully to quenching surface-boundfluorescein (Nichols et al. (1993) Arch. Virol. 130, 441; Collins &Buchholz (2005) J. Virol. Meth. 128, 192-197). It was found, however,that these non-specific quenchers were less effective in quenching thefluorescence of phycoerythrin (PE), likely because some of thephycobilin fluorescent clusters of PE are buried deep in the proteinglobule and inaccessible to the occasional contacts with quenchingmolecules.

To achieve more efficient quenching, an “addressed” affinity quencherwas prepared, based on anti-PE antibodies. Specifically, fluorescencequenching dyes QSY-9 and QSY-21 (Invitrogen) were linked to a goatanti-R-phycoerythrin antibody (Rockland Immunochemicals # 600-101-387).Such an affinity quencher binds specifically to the PE tags labeling thevirus. The quenching occurs through Forster Resonance Energy Transfer(FRET) between the adjacent QSY molecules and phycobilin fluorescentclusters of PE rather than via direct contacts of the fluorochrome witha non-specific quencher.

To assess the quenching protocol, an interim model was used: MDCK cellstagged with biotinylated anti-LDL antibodies and stained with SA-PEconjugate. This model mimicked the same MDCK cells bearingsurface-attached PE-labeled virus. The anti-PE quenching QSY-9-linkedantibody was added to the labeled cells at 10-30 μg/mL, and the cellswere incubated at 4° C. Combined with 400 μg/mL trypan blue, anti-PEquenchers subdued 65% of the PE fluorescence of the labeled cells (FIG.4).

Example 4 Use of a BioPlex Bead Array Reader as a Simplified FlowCytometer

Upon incubation of fluorescently labeled virus with target cells, thelatter can be read in a flow cytometry device to detect incorporatedvirus. Modern flow cytometers are versatile and expensive machines ableto monitor multi-colored labels. This powerful capacity may be excessiveand too expensive for microneutralization assays, which are intended tobe high-throughput and routine procedures.

In this example, we used a BioPlex-100 bead array reader for reading PEfluorescence of the virus engulfed by the target cells (fmNt mode) orattached to the surface of the target cells (fADI mode). The BioPlexreader and its accessories are significantly less expensive than a flowcytometer, and the reader requires less maintenance. Literatureregarding BioPlex readers may be found at the website beginning with“www.” and ending with“bio-rad.com/cmc_upload/Literature/54967/Bulletin_(—)2890.pdf.”

The BioPlex reader is designed for immunosorption experiments, where itreads fluorescence from phycoerythrin-conjugated anti-analyte antibodiesattached to the 5.6-μm plastic beads coated with analyte-capturingantibodies. The beads flow through the capillary fluorescence chamber.

In general, this scheme is similar to a most basic flow cytometryexperiment, where calibrated micro beads play the role of the cells.However, there is an important difference. The beads used in the BioPlexare coded via their intrinsic red/infrared fluorescence in such a waythat the ratio of the red and infrared components determines the codenumber of the bead. This allows discrimination of signals from amultitude (currently, up to 200) of beads with different codes, whichprovides for the exceptional multiplexing capacity of the BioPlex(BioRad Bulletin 2890).

As a result of this design, fluorescence signals from ordinary cellscannot be measured on the BioPlex directly, because the cells do notproduce a recognizable red/infrared fluorescence code. However,additional staining of the cells with an appropriate red/infraredfluorescent dye(s) can overcome this hurdle. We found that commonstaining dyes, such as trypan blue (TB) and crystal violet (CV), whenused at low concentrations, were able to ‘code’ target cells such asMDCK and Vero mammalian kidney epithelial cultures, or avianerythrocytes. TB, a dark blue staining dye, is commonly used in the dyeexclusion method, where apoptotic cells with damaged surface membranestake up the dye, turning dark blue, while normal cells do not. However,it is also true that normal cells become weakly stained with TB on theirsurface, which is normally not observable by light microscopy as used inthe dye exclusion assays. TB possesses a weak red/infrared fluorescence,and surface staining of normal and healthy cells with TB occurssufficient for their recognition by the classification system of theBioPlex reader as readable objects. Damaged and dying cells carry higherloads of TB, which results in stronger red/infrared fluorescence and,accordingly, different positioning of the cells in the classificationpanel of the BioPlex. This allows effective discrimination betweennormal and abnormal cells in these flow experiments (FIG. 5).

It was found that staining cells with TB usually produced certainbackground fluorescence in the detecting channel of the BioPlex reader,which is tuned to the orange fluorescence of the PE fluorochrome.However, this background was normally significantly lower than thesignal from the PE tag of the labeled viruses in and on the targetcells, and therefore the background and the informative signal from theSA-PE tag could be readily discriminated (FIGS. 5 and 6).

Example 5 Comparison of Results Obtained in the Flow Cytometer and theBioPlex Bead Array Reader

To demonstrate the capacity of the BioPlex to function as a simplifiedflow cytometer, an experiment on quenching of the PE fluorescence ofsurface-labeled MDCK cells was performed using a BD LSR II flowcytometer (PE channel) and a BioPlex-100 bead array reader in parallel(FIG. 6).

The BioPlex results were found to be fully consistent with the flowcytometer data. The efficiency of cell reading in the BioPlex was about35-40%. The BioPlex also showed a capacity to produce reliable resultsfrom a limited number of the registered cells as compared to tens ofthousands cells normally required for the experiments on a flowcytometer. In the conditions of the experiment, reading regions #11, #17and #25 were selected for calculating the pool averaged meanfluorescence index (MFI), which represented the averaged fluorescencesignal from the target cells.

Example 6 BioPlex-Based fmNt Assay

Two identical 96-well format round-bottom plates were prepared as shownin the layout table in FIG. 7. In these plates, MDCK target cells weremixed with influenza H1N1 Solomon Islands BPL-inactivated virus,affinity-labeled using anti-influenza A biotinylated antibody and SA-PEconjugate. The labeled virus was, or was not pre-incubated withhigh-titer anti-influenza human serum. One of the plates was furtherincubated at 37° C., where endocytosis of the virus is efficient.Another plate was incubated at 4° C., where endocytosis is stronglysubdued. After the incubation, the plates were centrifuged (400 g; 4°C.) and washed with cold PBS solution twice. Then the cells in half ofthe wells in each plate were re-suspended with 0.1% BSA in cold PBS,while the cells in the other half were re-suspended in the same solutioncontaining 30 μg/mL anti-PE:QSY quencher, and the plates were incubatedfor another 1 h at 4° C. Then, 70 μL of PBS containing 160 μg/mL trypanblue was added to each well, the plates were incubated for min at roomtemperature and read in the BioPlex reader. After that, another portionof 30 μL of concentrated TB solution was added, to a final concentrationof 600 μg/mL, and the plates were read again (see FIG. 7 for theprotocol and FIGS. 8 and 9 for the results). The resultant meanfluorescence index (MFI) numbers were calculated using the same readingregions #11, #17 and #25, and the same calculation procedure asdescribed in Example 5.

The results of the BP-fmNt experiment shown in FIGS. 8 and 9 demonstrateimportant features of the fmNt assays of the present invention. Withoutvirus, neither SA-PE conjugate alone, nor its complex with biotinylatedanti-influenza antibody produced any significant fluorescence in thetarget cells, showing only a low non-specific background coming from TBstaining of the target cells. In contrast, influenza virus labeled withthe anti-influenza:biotin:SA-PE complex and applied to the target cellsprovided bright fluorescence. This demonstrated the efficientinteraction of the target cells with the labeled virus. Pre-incubationof the labeled virus with human anti-influenza serum significantlyreduced the fluorescence of the target cells, as it should be expectedin the neutralization experiment. This reduction was more dramatic forthe samples incubated at 37° C. versus 4° C. The latter effect likelyreflected the relatively smaller portion of the labeled virus bound tothe surface of the target cells at 37° C., compared with engulfed virus.The quenching effect of both the PE-specific quenching antibodies andnonspecific TB was stronger for samples incubated at 4° C. versus 37° C.This likely reflected the higher share of the surface-bound virustowards the engulfed virus, as it should be expected at 4° C., whereendocytosis is effectively subdued.

In summary, the BP-fmNt assay described in this example demonstratedcrucially important elements of a microneutralization experiment: (i)selective binding of the labeled virus by the target cells, but not ofthe bare label; (ii) efficient quenching of the surface boundfluorescence, and (iii) efficient blocking of virus attachment andengulfment by anti-virus serum.

Example 7 Determination of the Neutralizing Titer of CommercialAnti-Influenza Polyclonal Antibodies Using the fmNt Technique

The neutralizing capacity of commercial anti-influenza A polyclonalantibodies was determined using the fmNt protocol described in Example6, with the following minor modifications. The concentration of thelabeled virus was reduced; the second reading with an increasedconcentration of TB was eliminated, and dilutions of the testedantibodies varied from 100 to 72,900 (FIGS. 10, 11). All the sampleswere assayed in duplicates. The dilutions corresponding to the 50%cut-off of the fluorescence, chosen as the neutralizing titers, weredetermined by the least-square best fit to the theoretical titrationcurve. The fmNt titers, 520 and 350, obtained for the commercialpolyclonal antibodies, ViroStat #1031 and Millipore # ab1074,respectively (FIG. 11), corresponded to concentrations of the IgG of70-90 nM, demonstrating that these antibodies were weak-to-moderateneutralizers.

Example 8 Determination of the fmNt Titers of Human Sera

The neutralizing capacity of human anti-influenza sera was assessedusing a scheme similar to that used for the commercial anti-influenzaantibodies described in Example 7. Samples of human sera taken beforeand after vaccination from donors #355 and #419 (high-level responders,as was found in earlier screening of the sera samples in the standardHAI assays) were pre-diluted roughly in accordance with their expectedneutralizing capacity, and then serially diluted as shown in FIG. 12,which displays the results of the assay. The fmNt experiments showed asignificant increase of the neutralizing titers of the post-vaccinationversus pre-vaccination sera. The fmNt titers demonstrated that theneutralizing capacity of the anti-influenza antibodies of thepost-vaccination human sera was ˜100 times higher than for thecommercial antibodies, taking into consideration an average IgG level˜10-15 mg/mL in the normalized human sera, ˜10% of which can be ascribedto an anti-influenza immune response. For example, the fmNt titer forthe post-vaccination serum #355 was determined to be ˜15000,corresponding to a concentration of neutralizing IgG of ˜0.7 nM (comparewith the results for the anti-influenza A antibodies in Example 7, FIG.11).

Example 9 Comparison of Neutralizing Titers Found in the fmNt Assayswith BPL-Inactivated Virus with Approved MN Protocols Using Live Virus

Comparative microneutralization experiments were performed on a panel of16 sera from eight donors vaccinated in the 2007/2008 flu season. Thesera were selected from a whole panel of 36 sera, in such a way thattheir HAI titers would cover a wide range, from the lowest titers forthe pre-vaccination sera to the highest titers of high-respondingpost-vaccination sera.

The fluorescent microneutralization (fmNt) experiments using theBPL-inactivated Solomon Islands H1N1 virus were performed in March-April2009.

Solomon Islands H1N1 virus was expanded on the MDCK culture, andmicroneutralization (MN) assays using the immunosorption enzyme linkedprotocol (CDC protocol; Rowe et al. (1999) J. Clin. Microbiol. 37,937-943) and direct MN protocol based on hemagglutination (HA)measurements of the expanding virus (WHO protocol described in the “WHOManual on Animal Influenza Diagnosis and Surveillance,”(WHO/CDS/CSR/NCS/2002.5 Rev. 1) using live Solomon Islands H1N1 viruswere performed in May-August 2009.

The results shown in FIG. 13 demonstrate remarkable parallelism in theneutralization titers obtained for the inactivated virus using the fmNttechnique, and for the live virus using the standard CDC and WHOprotocols. This observation was corroborated by significantcross-correlation coefficients for the inactivated virus and the livevirus results, shown in Table 1.

TABLE 1 fmNt FIA-CDC MN-WHO fmNt X 0.901 0.661 FIA-CDC X X 0.904 MN-WHOX X XAdditionally, the fmNt assay demonstrated sensitivity to theneutralizing sera 3-5 times higher than the FIA and the MN protocols, ascan be seen by comparing the corresponding MN and fmNt titers for thedifferent assays in FIG. 13.

Example 10 Fluorescent Adherence Inhibition Assay (fADI)

Adherence of the virus to the surface of the target cells is normallyconsidered an obstructing factor in fluorescent microneutralization,which should be minimized or eliminated. However, surface adherence ofthe virus is a necessary step for infection, preceding engulfment by thetarget cell. Logically, such a phenomenon has no less relevance toinfectivity of the virus than agglutination of erythrocytes employed assignaling factor in the HA and HAI assays. It is reasonable to expectthat virus-specific antibodies will be able to block surface adherencewith an efficiency at least comparable with that demonstrated inblocking the penetration of the virus into the target cells.

The well-known and widely used hemagglutination inhibition assay (HAI)actually explores blocking of the attachment of the virus to the surfaceof the target cells (erythrocytes). The importance of the HAI andcontinuing interest in using it supports the idea that monitoring of theinhibition of adherence of the virus to the target cells by avirus-specific antibody, in general, can provide data of significantinterest. Further, the protocol for a fluorescent adherence inhibitionassay (fADI) can be simpler, shorter, and less material- andtime-consuming than a fmNt experiment, because the fADI does not requireapplication of surface fluorescence quenchers and an additionalincubation (FIG. 14). The fADI assay measures the capacity ofvirus-specific antibody or sera to block adherence of the virus to thetarget cell. Importantly, the fluorescent fADI experiment can provide astronger fluorescence signal from the target cells (FIG. 15). This, inits turn, can allow working at lower concentrations of the labeledvirus, thus providing higher sensitivity of the assay versus the fmNt,as is shown in the titration of a commercial anti-influenza A antibody(ViroStat #1301) using fmNt and fADI methods, displayed in FIG. 16. Thedilutions of the affinity-labeled influenza virus used in these assayswere 100 and 3200, respectively, and the 50% blocking titers for theantibody were found as 520±180 and 32700±18500 (i.e., roughlyproportional to the virus dilution).

Example 11 fADI Experiments Using Turkey Erythrocytes and New CaledoniaH1N1 Influenza Virus

The capacity of influenza viruses to attach to erythrocytes of differentspecies (e.g., human, guinea pig, swine, chicken, turkey) is widely usedin the routine titration of virus cultures in the HA technique, and intesting the neutralizing capacity of sera with HAI assays. In thesemethods, erythrocytes used at relatively high concentrations (˜1% HCT)are agglutinated by virus particles in a translucent three-dimensionalgel matrix. At lower concentrations, spatial agglutination is notpossible, although virus attachment remains strong. Adherence of thefluorescently labeled virus to erythrocytes can be detected in a flowcytometry experiment analogous to that described above for MDCK targetcells.

For such experiments, large and heavy avian erythrocytes were found tobe preferable to mammalian erythrocytes, because the BioPlex bead arrayreader and the flow cytometer better detected the former.

Fresh samples of turkey blood balanced with citrate buffer were washedthree times in PBS (at 400 g), and the upper layer of the pelletcontaining lymphocytes was discarded. The washed erythrocytes werediluted in 1% human serum albumin (HSA) in PBS to the level of 0.03%HCT.

As an example, New Caledonia H1N1 BPL-inactivated virus was chosen forthese experiments (Solomon Islands H1N1 strain also showed acceptableresults; data not shown). New Caledonia H1N1 BPL-inactivated virus,affinity labeled with the same biotinylated anti-H1 ViroStat antibody#1307, as described above for Solomon Islands H1N1, was finally diluted3200-fold in 1% HSA in PBS. Samples of donor sera were diluted in 1%HSA/PBS to the levels of their previously determined HAI titers (e.g.,the sample of the post-vaccine serum #608 with a HAI titer of 320 wasdiluted 320-fold), and then subjected to a further 10-fold sub-dilutionfollowed by the two-step triple serial sub-dilution (e.g., to 1 to 3200,1 to 9600, and 1 to 28800, for the post-vaccination serum #608). Thealiquots of diluted sera and labeled virus, 40 μL of each, were mixed in96-well round bottom plates and incubated in the refrigerator (˜4° C.)for 40 min. Then, 40-μt aliquots of diluted erythrocytes were added,thus making the final volumes 120 μL, and the plates were incubated foranother 30 min on the bench at room temperature on an XY shaker with lowshaking (˜500 rpm). Then, the plates were centrifuged (400 g, 4 min),and the supernatant was discarded. The erythrocyte pellets werere-suspended in 120 μL of 1% HSA/PBS per well, and centrifuged again.The next wash was performed using 1% HSA/PBS containing 4.5 μg of TB, tostain erythrocytes and make them suitable for classification in theBioPlex bead array reader, in the manner described in Example 4 for MDCKcells. With this low-level staining, the classification region #1 of thebead array reader was used for reading the TB-stained erythrocytes.After the last wash, the erythrocytes were resuspended in the samestaining solution, 100 μL per well, and the plate was read in theBioPlex reader. FIG. 17 shows typical results of the fADI titrations forthe pre- and post-vaccination sera of donors #608 and #145. The fADItitration curves were fit to the standard sigmoidal titration curve todetermine fADI subtiters, as described in Examples 7 and 8. The finalfADI titers were found as products of the initial serum dilution and thefound fADI sub-titer (e.g., for the post-vaccine serum #608, the finalfADI titer was 320×26.7=8550.4).

The fADI experiments with affinity-labeled New Caledonia H1N1 influenzavirus and turkey erythrocytes described here were further performed fora panel of 36 pre- and post-vaccine donor sera. FIG. 18 demonstrates agood correlation between the classical HAI and fADI data, as well as anapproximately 80-fold higher sensitivity of the fADI technique, as shownby the slope of the scatter plot.

Example 12 fADI Experiments Using Turkey Erythrocytes and Wisconsin H3N2Influenza Virus

After successful testing with the H1N1 virus, the fADI technique withturkey erythrocytes was also examined with the H3N2 virus. The affinityfluorescent labeling was performed basically as described in Example 1and Example 11 for Solomon Islands and New Caledonia H1N1 viruses, butusing ViroStat anti-H3 biotinylated antibody #1317 instead of theanti-H1 #1307. Also, the final dilution of the virus was 400-fold ratherthan 3200-fold for the New Caledonia H1N1 virus, due to the weakercapacity of the Wisconsin H3N2 strain to adhere to target cells. Samplesof the donor sera were diluted in 2% BSA/PBS to the levels of theirpreviously determined HAI titers for the Wisconsin strain (e.g., thesample of the post-vaccination serum #608 having the HAI titer of 160was diluted 160-fold), and then subjected to a further 20-foldsub-dilution, followed by two-step triple serial sub-dilution (e.g., to1 to 3200, 1 to 9600 and 1 to 28800, for the post-vaccination serum#608). The rest of the experimental protocol was similar to thatdescribed in Example 11 above. FIG. 19 shows typical results of the fADItitrations for the pre- and post-vaccine sera of the donor #608.

The fADI experiments with affinity-labeled Wisconsin H3N2BPL-inactivated influenza virus and turkey erythrocytes were furtherperformed for a panel of 36 pre- and post-vaccination donor sera. FIG.20 demonstrates the correlation between the HAI and fADI data, as wellas an approximately 70-fold increased sensitivity of the fADI technique,compared with the classical HAI. Higher scattering of the data in thefADI versus HAI plot can be explained by altogether weaker binding andagglutinating capacity of the H3N2 virus, which resulted in lessreliable titration in the traditional HAI assay.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts, and other references cited herein areincorporated by reference in their entirety. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

1. A method for determining a neutralizing activity of a test antibody,comprising: a) incubating a test antibody with a fluorescently-labeledvirus to form a mixture, b) incubating a population of target cells withthe mixture of a) under conditions permitting endocytosis of the labeledvirus by the target cells, c) measuring fluorescence of labeled virusendocytozed by the target cells, and d) comparing the fluorescencemeasured in c) with fluorescence of fluorescently-labeled virus measuredin a control experiment where the labeled virus was not incubated with atest antibody, thereby determining a neutralizing activity of a testantibody.
 2. A method for determining a neutralizing activity of a testantibody, comprising: a) incubating a test antibody with afluorescently-labeled virus to form a mixture, b) incubating apopulation of target cells with the mixture of a) under conditionspermitting endocytosis of the labeled virus by the target cells, c)incubating the population of target cells of b) with a quencher offluorescence of the labeled virus bound to the surface of the cells, d)measuring fluorescence of labeled virus endocytozed by the target cells,and e) comparing the fluorescence measured in d) with fluorescence offluorescently-labeled virus measured in a control experiment where thelabeled virus was not incubated with a test antibody, therebydetermining a neutralizing activity of a test antibody.
 3. A method fordetermining a neutralizing activity of a test antibody, comprising: a)incubating a test antibody with a fluorescently-labeled virus to form amixture, b) incubating a population of target cells with the mixture ofa) under conditions permitting endocytosis of the labeled virus by thetarget cells, c) incubating the population of target cells of b) with aquencher of fluorescence of the labeled virus bound to the surface ofthe cells, d) staining the population of target cells of c) with a dye,e) measuring fluorescence of labeled virus endocytozed by the targetcells, and f) comparing the fluorescence measured in e) withfluorescence of fluorescently-labeled virus measured in a controlexperiment where the labeled virus was not incubated with a testantibody, thereby determining a neutralizing activity of a testantibody.
 4. A method for determining an inhibitory activity of a testantibody, comprising: a) incubating a test antibody with afluorescently-labeled virus to form a mixture, b) incubating apopulation of target cells with the mixture of a) under conditionssubduing endocytosis and permitting cell surface adherence of thelabeled virus to the target cells, c) measuring fluorescence of labeledvirus adhered to the surface of the target cells, and d) comparing thefluorescence measured in c) with fluorescence of fluorescently-labeledvirus measured in a control experiment where the labeled virus was notincubated with a test antibody, thereby determining an inhibitoryactivity of a test antibody.
 5. A method for determining an inhibitoryactivity of a test antibody, comprising: a) incubating a test antibodywith a fluorescently-labeled virus to form a mixture, b) incubating apopulation of target cells with the mixture of a) under conditionssubduing endocytosis and permitting cell surface adherence of thelabeled virus to the target cells, c) staining the population of targetcells of b) with a dye, d) measuring fluorescence of labeled virusadhered to the surface of the target cells, and e) comparing thefluorescence measured in d) with fluorescence of fluorescently-labeledvirus measured in a control experiment where the labeled virus was notincubated with a test antibody, thereby determining an inhibitoryactivity of a test antibody.
 6. The method of claim 2 or 3, wherein thequencher of fluorescence of labeled virus bound to the surface of thecells is an antibody that specifically binds the fluorescent label ofthe labeled virus and wherein the antibody is conjugated to at least onequenching compound.
 7. The method of claim 6, wherein the quenchingcompound conjugated to the antibody is a quenching dye.
 8. The method ofclaim 7, wherein the quenching dye is quenching dye QSY-9 or quenchingdye QSY-21.
 9. The method of claim 3 or 5, wherein the dye staining thepopulation of target cells is a dye that has a weak red and infraredfluorescence, thereby facilitating classification of the target cells ina bead array reader, and that quenches the fluorescent label of thelabeled virus.
 10. The method of claim 9, wherein the dye is trypan blueor crystal violet.
 11. The method of claim 1, wherein the labeled viruscomprises a virus selected from the group consisting of influenzaviruses, adenoviruses, filoviruses, flaviviruses, herpesviruses,poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses,orthomyxoviruses, rhabdoviruses, retroviruses, and hepadnaviruses,conjugated to a fluorescent label.
 12. The method of claim 1, whereinthe labeled virus comprises a biotinylated virus selected from the groupconsisting of influenza viruses, adenoviruses, filoviruses,flaviviruses, herpesviruses, poxviruses, parvoviruses, reoviruses,picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses,retroviruses, and hepadnaviruses, conjugated to astreptavadin:fluorescent label.
 13. The method of claim 1, wherein thelabeled virus comprises a biotinylated virus selected from the groupconsisting of H1N1 influenza virus, H3N2 influenza virus, H5N1 influenzavirus, Marburg hemorrhagic fever virus-like particles, andgamma-inactivated Ebola virus, conjugated to a streptavadin:fluorescentlabel.
 14. The method of claim 1, wherein the virus is an inactivated oran attenuated virus.
 15. The method of claim 1 wherein the virus is avirus inactivated using betapropiolactone (BPL) or UV or gammairradiation.
 16. The method of claim 1, where the population of targetcells comprises a human cell line or avian erythrocytes.
 17. The methodof claim 1, where the population of target cells comprises Madin-Darbycanine kidney epithelial cells or Vero green monkey kidney epithelialcells.
 18. The method of claim 1, wherein the measuring is detecting thelabeled virus using a flow cytometer or a bead array reader.
 19. Themethod of claim 1, wherein the neutralizing activity of sera or anantibody is blocking entry of the labeled virus into the target cells.20. The method of claim 4, wherein the blocking activity of sera or anantibody blocking adherence of the labeled virus to the target cells, orblocking entry of the labeled virus into the target cells.